The advent of RNA interference as a mechanism for post-transcriptional silencing of gene function in mammalian cells represents a quantum advance and tremendous opportunity for experimental analysis of gene function (Hannon and Rossi, 2004;Novina and Sharp, 2004). Remarkable progress has been made over the past several years in harnessing the RNAi pathway for facile genetic manipulation of cells and animals. With the ongoing development of genome-wide libraries of siRNAs and shRNAs for multiple species, we have entered a new era in which the application of powerful forward genetic approaches can be used both in vivo and in vitro to assign function to genes, delineate molecular pathways in which these genes affect normal and disease cellular processes, and to contribute to the knowledge necessary to develop new and improve existing therapies. The Duke Center for RNA Biology, the Duke Comprehensive Cancer Center, and the Center for Applied Genomics and Technology, a component of the Duke Institute for Genome Sciences and Policy, have come together to establish an RNAi shared-resource facility. A primary mission of the RNAi Facility is to develop and deploy RNAi technologies to support functional genomics research programs at Duke. Through large investments in RNAi and complementary technologies, we are assembling a state-of-the-art infrastructure for functional genomics, providing researchers access to genome-wide RNAi reagents and the infrastructure necessary to conduct large-scale loss-of-function studies in mammalian cells. In this proposal, we seek support to significantly enhance this technology infrastructure with the acquisition of the Molecular Devices IsoCyte Laser Scanning Plate Cytometer for high-throughput, high-content screening (HT-HCS). The HT-HCS capabilities of the Isocyte combine the power of high-content cell-based assays with true high-throughput workflow. The HCS technology of the IsoCyte enables ultra-high-throughput workflows when using traditional cell-based assays, while providing the ability to assay large multicellular organisms like C. elegans and zebrafish. In addition, the unique architecture and scanning speed of this instrument allows interaction studies to be performed in real-time, in individual cells using Fvrster Resonance Energy Transfer (FRET) by anisotropy. When coupled with upstream RNAi assay automation and downstream informatics, HT- HCS represents a tremendous technological advance, enabling researchers to extract and quantify useful biological information, and to identify genes with roles in virtually and cellular processes, including disease.